Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15

Bacteria of the genus Aeromonas, especially A. hydrophila and A. veronii are recognized as important fish pathogens that cause significant economic losses in aquaculture. Environmentally friendly bacteriophage-based solutions for the treatment of fish and for the reduction of colonization by pathogenic bacteria in production facilities are currently in high demand. The bacteriophage Gekk3-15 was isolated during a search for novel phage strains potentially suitable for Aeromonas biocontrol applications. Genome sequencing revealed that this virus is a relatively small myovirus with a 64847 bp long dsDNA genome, which is consistent with virion electron microscopy data. Bacteriophage Gekk3-15 is distinct in its nucleotide and encoded aa sequences from all other sequenced bacteriophage genomes, and may represent a new viral taxon at the genus or subfamily level.


Introduction
Currently, aquaculture is the primary producer of nutritional fish in most developed countries. 1The cultivation of hydrobionts in production ponds is associated with very high densities of animal populations, which far exceed the typical population densities in wild habitats.Such conditions make cultivated fish specifically vulnerable to bacterial infections, of which aeromonoses comprise a significant fraction. 2,3The use of antibiotics to reduce economic losses associated with bacterial infections is restricted or prohibited in many countries; therefore, using phages as a tool for environmentally friendly biocontrol of the iсhtyopathogenic Aeromonas has attracted significant interest in scientific and business communities.Therefore, the search for new bacteriophage strains that are potentially suitable for use as phage therapy agents is important.Here, we present the isolation and sequencing of a novel A. veronii bacteriophage that represents a new group of bacteriophages that infect the genus Aeromonas.

Phage isolation and cultivation
The bacteriophage Gekk3-15 was isolated as part of a project to establish a panel of bacteriophages to target the collection of A. hydrophila and A. veronii strains that cause infections in aquaculture facilities in Russia.The phage was isolated from a water sample collected from the Norishka Stream in Mikhailovsky Park in Moscow, Russia, on September 14, 2021.The bacteriophage was isolated using the enrichment culture setup as follows: A non-sterilized water sample (0.5 l) was placed in a 2 l sterile plastic bottle and supplemented with 50 ml of sterile 10x LB medium (for 1Â medium: 10 g Bacto Tryptone (Amresco, Am-J859-0.25),5 g yeast extract (Amresco, J850-500G), 10 g NaCl (Amresco, J869-500G), distilled H 2 O up to 1 l).The culture was supplemented with 0.5 ml of an overnight liquid culture of A. veronii AV-3 strain and incubated at 28 °C in a thermostat without agitation.Following incubation, 1 mL of the enrichment culture was centrifuged at 12 000 rpm in a tabletop microcentrifuge, filter-sterilized using a 0.22 μm syringe membrane filter (Millex-GP, Millipore), and plated onto the lawn of A. veronii strain 4 using the conventional double-layer technique. 4age plaques were formed after an overnight incubation at 28°C.The phage was purified using two consecutive singleplaque isolations.To obtain a high-titer lysate, five 90 mm Petri dishes were filled with solid LB medium (15 g of Bacto Agar (BD214030, BD Difco) per 1 l).Without drying the plates, they were overlaid with 5 ml of soft LB agar (6 g Bacto Agar (BD214030, BD Difco) per 1 l) per plate.Soft agar was inoculated with 300 μl of a 4h log-phase liquid culture of the host strain and approximately 1Â10 5 PFU of the phage per plate.The plates were incubated overnight at 28 °C.To extract the phage, the soft agar layer was gently destroyed with a spreader, transferred into 50 ml plastic centrifuge tubes, and layered with 10 ml of LB medium.Chloroform (Fisher Scientific, C298-4)(100 μL) was added to each tube, followed by vigorous vortexing for 1 min, and allowed to stand at room temperature for 2 h.After incubation, the agar fragments and bacterial cells were pelleted by centrifugation at 10 000 g for 5 min, and the supernatant was collected and centrifuged again under the same conditions.The phage was purified using a sucrose gradient as it described elsewhere. 5The purified phage sample was used for DNA extraction and transmission electron microscopy (TEM), as previously described. 6A extraction, sequencing, assembly and annotation To extract DNA, the phage stock with a titer of approximately 10 11 PFU ml À1 was treated with DNAse (Thermo Scientific, EN0521)(0,01 mg ml À1 ) for 30 min at room temperature, and the phage particles were then collected by ultracentrifugation in an angle rotor at room temperature (Beckman 45Ti, 1 h, 75000 g).Genomic DNA was extracted from the precipitates with CTAB cetyltrimethylammonium bromide (CTAB) extraction as described previously.7 DNA quality and quantity were assessed using agarose gel electrophoresis and a Qubit dsDNA HS fluorometer assay (Qubit, USA).Phage genomic DNA libraries were prepared and sequenced using an Ion Proton sequencer (Applied Biosystems, Foster City, CA, USA) with the standard chemistry according to the manufacturer's instructions.The raw reads from the run were combined and filtered using the error correction tool Pollux (https://github.com/emarinier/pollux). Cotigs were assembled using Newbler version 3.0 (RRID:SCR_011916) (Roche Diagnostics, USA).A single contig with a phage genome of 64 847 b.p. with an average coverage of 200 bp was obtained.

Results
The bacteriophage Gekk3-15 was found to be a relatively small myovirus with an isometric head (Figure 1).The genome consisted of 64 847 b.p. and encoded 101 ORF, including putative virion structural proteins.A set of proteins for the contractile phage tail was detected, confirming the bacteriophage TEM examination data.The Gekk3-15 genome also contains genes for DNA metabolism enzymes, a cell lysis system, and two potential auxiliary metabolic genes (AMGs), encoding 3-oxoacyl-[acyl-carrier-protein] reductase FabG and glyoxylase/dioxygenase superfamily proteins.No tRNA genes were identified.Phage Gekk3-15 does not contain any markers potentially associated with a lysogenic lifestyle or virulence factors.A BLASTN (RRID:SCR_001598) search against the NCBI nucleotide collection did not produce any relevant hits for related bacteriophage genomes.However, the BLASTX (RRID:SCR_001653) search identified a number of distantly related viruses, among which the closest relative by the large terminase a.a.sequence was the Pseudomonas phage EPa61 (GenBank NC_048744), sharing 57% aa identity with the Gekk3-15 protein.The levels of a. a. identity between the structural proteins of bacteriophages Gekk3-15 and EPa61 varied between 38% and 63%.This degree of similarity indicates that taxonomically, these phages belong to different genera and, probably, different subfamilies within the Caudoviricetes kingdom.Therefore, we conclude that phage Gekk3-15 represents a novel genus and, probably, a novel bacteriophage taxon of a higher rank.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 17 June 2024 https://doi.org/10.5256/f1000research.157743.r278893

Major comments:
The framing of the study may be too broad for the content.The abstract discusses the use of phage in Aeromonas hydrophila, but this is not tested in the main text, and it is unclear if this particular phage would infect this host.We recommend either re-framing the abstract to specify Aeromonas veronii only, as this is the only isolate tested, or to test the phage infectivity on A. hydrophila also.This is especially important if the aim of the phage isolation is to be used in phage therapy. 1.

Minor comments:
Page 3, under Methods, 'Phage isolation and cultivation', paragraph 1: "at 28 deg.Celsius in a thermostat without agitation"."at 28 deg.Celsius in a thermostatic incubator without agitation" would be clearer.
Same paragraph: please add how many minutes the enrichment culture was centrifuged for.The x g should be listed rather than rpm Same paragraph: it is unclear what A. veronii strain was used to isolate this phage, initially it is listed as AV-3 strain, and later as strain 4. Either these are different strains or one was mis-named in the manuscript, but regardless it would be pertinent to introduce any strains used and include information about the origin(s).
Page 3, under Methods, 'Phage isolation and cultivation', paragraph 2: what method was used for single-plaque isolations?Please provide a reference.
Same paragraph: should read 'as described elsewhere' not 'as it described'.
The authors attempt to classify their phage into an appropriate taxonomy based on amino acid identity of structural proteins.While this is not the gold standard as recommended by the ICTV (nucleotide similarity across the genome as specified by VIRIDIC), it is probably as close as is possible, given the paucity of suitably similar reference genomes.For instance we attempted to identify the closest phage genome in both INPHARED (Cook et al., 2021 1 ) and PhageClouds (Rangel-Pineros et al., 2021 2 ) (at both 0.15 and 0.2 distance), which yielded no hits.Therefore, the cautious interpretation of the placement by the authors is appropriate and should be revisited when more closely related genomes are deposited.

provided to allow replication by others? Partly
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?Yes Competing Interests: No competing interests were disclosed.

Reviewer Expertise: phage biology; phage bioinformatics
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Introduction Reference
No. 1 could be replaced by the FAO report or another article more relevant to the statement."Aeromonoses" seems to be a term not globally used.Replace it with "Aeromonas infections" or " Aeromonas diseases."Bacteriophage isolates Â How many are the isolates from the collection of the two Aeromonas species?LB, the full name of the medium should be provided before the abbreviation.
How was it identified that AV-3 corresponds to the species A. veronii?This should be assured, as the taxonomy of this group is not easy and requires molecular analysis.Reviewer Expertise: Some of my main research topics include pathology in aquaculture; diseases of aquatic organisms; biotechnological tools applied to aquaculture; standardization of procedures to control and validate the appropriate use of antibiotics; and the development of vaccines against aquatic pathogens.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Partly Competing Interests: No
Make this clarification for all Aeromonas used in this study."Strain"or"isolates," I think the latter.Review throughout the article.It is not necessary to give the LB recipe twice; just indicate the agar percentage in the last case.What concentration of DNA was sent for sequencing?This data is very important.Specify what "bp" is and correct the abbreviation, removing the periods.ResultsIndicate that it belongs to the family Myoviridae instead of "myovirus" in italics.The last lines where the conclusion is drawn, I believe it is too speculative to state that it corresponds to a new genus and further tests are required to confirm it.The article should modify the conclusion and reduce the enthusiasm, unless there are other tests that prove it.I suggest modifying it.If it was tested with a collection of Aeromonas, I would like to know if the activity is broad-spectrum or if it's only an isolate that was analyzed.This situation is very important.Additionally, was any physiological characterization of the virus conducted, considering that the introduction suggests they are an alternative for use in fish aquaculture?
competing interests were disclosed.